Historically, biomolecules labeled with radioactive isotopes or colorimetric dyes have been employed in bioanalytical procedures. The inherent disadvantages of radioactive labels (safety concerns, disposal costs, record keeping and often poor label stability) and low sensitivity of colorimetric dyes have led to increasing use of luminescent molecules as reporters. Besides having the potential of higher sensitivity than radioactive or colorimetric reporters, luminescent reporters also allow simultaneous detection of multiple analytes, improved stability, reduced costs, improved spatial resolution and greater scope for signal modulation, a feature which permits homogeneous assays to be designed.
Two types of luminescence methods have been employed in bioassays. They are chemiluminescence and fluorescence. Chemiluminescence refers to the transient emission of light during an enzymatic reaction. Fluorescence is the emission of longer wavelength (visible region) of light by a molecule when excited by shorter wavelength (usually in the UV region) of light. If the luminescence is generated by an enzyme, an extra level of amplification is added to the system. Although, chemiluminescence is often regarded as the most sensitive signal system, fluorescence has certain advantages over chemiluminescence. A chemiluminescent molecule can decay only once, giving a single photon, while a fluorophore is continuously excited, having the capacity to emit tens of thousands photons before losing fluorescence due to chemical degradation or photobleaching. Other disadvantages of chemiluminescence include: laborious requirements for use, limitations on sensitivity arising from the transience of chemiluminescence itself, and, the broad spectral emission which precludes the simultaneous detection of multiple analytes in a single sample.
At its limit, fluorescence can detect single molecules either by fluorescence correlation spectroscopy or other methods. However, in practice, the extreme sensitivity potential is limited by interfering signals (noise). These signals may originate from biological components, plastics, reagent impurities, light scattering from particles, background light (“stray” light) which exists in all fluorescence detectors because of the light source used to induce excitation, contamination from a variety of sources, poor aqueous solubility of fluorogenic molecules, significant photoquenching and poor photostability in aqueous solutions and low turnover rates of available fluorogenic substrates by their specific enzymes.
Frequently, in biochemical and immunochemical assays, it is the noise, rather than the instrumentation, that limits sensitivity. As a result, progress in fluorescence has largely been about obtaining selectivity, i.e. improving the signal-to noise ratio. Several techniques have been developed to avoid the noise problem. Time resolved fluorescence, fluorescence correlation spectroscopy and confocal microscopy largely eliminate noise problems since only fluorophores in solution are detected. There is also a trend towards the use of fluorophores that are excited and emit at longer wave lengths where noise sources are less of an issue. Signals from fluorophores with large Stokes' shifts are more easily distinguished from noise.
Unlike a chemiluminescent molecule, a fluorescent molecule can be derivatized so that it can be used either (i) to covalently attach as a fluorescent tag or (ii) as a “fluorogenic” enzyme substrate which can be converted by the action of an enzyme specific for that substrate, into a fluorescent product which exhibits greatly enhanced fluorescence as compared to the starting substrate. The rate or extent to which a substrate for that enzyme has been converted into a detectable chemiluminescent or fluorogenic final product is used to detect or measure the activity of the enzyme.
In biological assays, the activity of an enzyme is used to indirectly detect or measure the quantity of a complementary biological “target”. Hydrolytic enzymes such as alkaline phosphatase, β-galactosidase, β-glucuronidase and β-glucosidase have been widely used in conjunction with fluorogenic substrates. These enzymes act on substrate molecules which have been derivatized at hydroxyl moieties to create phosphoric acid, galactoside, glucuronide and glucoside substrates, respectively, and the parent fluorescent molecule. Derivatives of fluorescein, ATTOPHOS® dyes, and BODIPY® dyes are some of the commercially available dyes for this purpose.
U.S. Pat. No. 5,443,986 (Haughland et al.) discloses detecting the activity of enzymes and enzyme conjugates using substrates made from a class of fluorophores generally including quinazolines, benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines and phenanthradines.
Kauffman et. al. (Chemical Abstracts, 130: 174202, 1999) discloses p-transfer fluors as shifters for green-emitting scintillating fibers of multi-fiber lengths.
International Patent Application Publication No. WO 00/03034 (Conrad et al.) describes halo-pyrene-disulfonic acids and their derivatives as potential fluorogenic dyes for hydrolytic enzymes. These compounds are described as being highly water soluble, highly stable to non-enzymatic hydrolysis and rapidly converted to the parent fluorescent products (with long wave lengths of excitation and large Stokes' shifts) by the action of appropriate enzymes. This patent also reviews the state of the art as it applies to pyerene derivatves.
U.S. Pat. No. 5,424,440 (Klem et al.) describes several hydroxy benzothiazoles derivatized at the hydroxy group with protective groups that can be cleaved with esterases. The following advantages were described for these compounds: stability in aqueous environment; easily detectable above background interference; highly fluorescent in a variety of solvents; sufficient Stokes' shift; sensitive enough to detect alkaline phosphatase at 10 attomolar concentration; long life time; and some members could be excited with visible light. The fluorogenic phosphates were stable in water and could be hydrolyzed to fluorescent compounds. This patent includes a review of the state of art at the time it was filed.
U.S. Pat. No. 5,587,112 (Kauffman et al.) describes alkaline phosphate substrates derived from 2-benzazolyl dibenzofurans, 2-benzazolyl dibenzothiophenes and 2-benzazolyl carbazoles. These belong to a class of fluorescent compounds called “excited state intramolecular proton transfer” (ESIPT) fluors. The phenomenon of ESIPT fluorescence results in large Stokes' shifts. Solutions of the ESIPT 2-benzazole fluors claimed in the '112 patent absorb strongly in the UV to blue spectral region, i.e., about 420 nm or shorter, with absorption maxima in the UV indicating an extinction coefficient of about 37,000 or greater. These 2-benzazole fluors possess unusually strong, UV stimulated, proton transfer fluorescence in the visible spectral region, having a fluorescence emission peak at about 520 nm or longer at room temperature or above room temperature. These compounds also exhibit fluorescence quantum yields of 0.5 or greater at 300° K.
Molecular Probes, Inc. catalog, 6th Edition, 1996, page 117 lists ESIPT fluor, ELF-97(a quinazolinone) and describes its applications in in situ hybridization, cytological labeling, immunohistochemistry and endogenous phosphate detection. This compound and other quinazolinones, benzothiazoles, benzoxazoles, benzimidazoles, quinolines, indolines and phenanthridines are claimed in U.S. Pat. No. 5,443,986. The preferred fluorogenic substrates described in this patent are said to have the following properties:                a. generally soluble and non-fluorescent in water but capable of releasing a highly fluorescent solid product in an aqueous solution containing the fluorogenic substrate and the specific hydrolase;        b. an excitation maximum of greater than about 340 nm and a Stokes' shift of about 100 nm for the solid product; and        c. the fluorescent solid is highly resistant to photobleaching.        
Tae-Il Kim et al (Chem. Commun., 5895, 2009) described the synthesis of the phosphate derivative of 2-(2′-hydroxyphenyl) benzothiazole (HBT) as substrate for protein tyrosine phosphatase. HBT is a known ESIPT fluor with a large Stokes shift. However HBT has low quantum efficiency. The compounds claimed in this patent have high quantum efficiencies besides possessing large Stokes shifts, radiation, thermal and chemical stability and good solubility in a wide range of solvents.
As the state of the diagnostic assay art advances there is a continuing need for new fluorogenic dyes which can be activated enzymatically or photolytically and conjugates labeled with novel fluorescent molecules which are useful in diagnostic assays.